In a cartilage tissue, chondrocytes exist in the state of being embedded in the matrix. These chondrocytes can be separated from the matrix by treating the cartilage with an enzyme such as collagenase. Attempts have been made to utilize these separated chondrocytes in transplantation therapy, in particular, autotransplantation of chondrocytes for treating cartilage-related diseases. It has been experimentally confirmed that transplantation therapy with the use of this method is applicable to animals such as rabbits and cows from which a large amount of cells can be obtained (see, for example, Bentry, et al., Nature 230: 385-388 (1971), Green, Clin. Orthop. 124:237-250 (1977); Wakitani et al., J. Bone and Joint Surgery 71B: 74-80 (1989); Paige et al., Plastic and Reconstructive Surgery 96:1390-1398 (1995); and Paige et al., Plastic and Reconstructive Surgery 97:168-178 (1996)).
Attempts have been also made to culture human chondrocytes in, for example, articular cartilage, auricular cartilage and costal cartilage (Aulthouse et al., In Vitro Cellular & Developmental Biology 25: 659-668 (1989); Brittberg et al., The New England Journal of Medicine 331: 889-895 (1994); Ting et al., Annals of Plastic Surgery 40: 413-421 (1998); and Rodriguez et al., Plastic and Reconstructive Surgery 103:1111-1119 (1999)).
In humans, however, only a small amount of cartilage can be collected, therefore, only a small number of chondrocytes can be used at the initiation of the culture. Moreover, human chondrocytes can be minimally proliferated by the conventional methods and proliferated chondrocytes, if any are obtained, convert into fibroblasts having different characters. Thus, it is highly difficult to apply human chondrocytes to transplantation therapy in practice. Namely, there is a problem that although a large amount of normal chondrocytes is required for transplantation in humans, it has been impossible to obtain a sufficient amount of human chondrocytes using the currently available methods.
To overcome this problem, the present inventor proposed to quickly culture a large amount of human chondrocytes by co-culturing human chondrocytes together with perichondral cells in the chondrogenic stage serving as feeder cells supporting the proliferation ability of the chondrocytes (WO 02/12451 (2002)). The use of nonhuman animal feeder cells is, however, accompanied with problems of unexpected bacterial or viral infections and complicated treatments are needed to prevent these infections.